Gaucher disease is an autosomal recessive disorder caused by a deficiency of glucocerebrosidase, the enzyme that is required for the lysosomal degradation of lipids containing covalently bound sugars (glycolipids). Brady et al., J. Biol. Chem., 240:39-43 (1965). In the absence of glucocerebrosidase, the extremely insoluble glucosylceramide (glucocerebroside) accumulates.
The gene for glucocerebrosidase is located on chromosome-1 in the region of q21. See, Shafit-Zagardo et al., Am. J. Hum Genet., 33:564-575 (1981); Ginns et al., Proc. Natl. Acad. Sci., U.S.A., 82:7101-7105 (1985). The fact that a number of different mutations caused Gaucher disease was inferred from clinical observations (Beutler, Genetic Diseases Among Ashkenazi Jews, eds. Boudman et al., Raven Press, NY, pp. 157-169 (1979)) and from differences in the kinetic properties of the residual enzyme in different patients with the disorder. Grabowski et al., Am J. Hum. Genet., 37:499-510 (1985). However, real understanding of the genetics of this disease has had to await the cloning and sequencing of the cDNA (Sorge et al., Proc. Natl. Acad. Sci., U.S.A., 82:7289-7293 (1985) and Tsuji et al., N. Engl. J. Med., 316:570-621 (1987)) and of the gene (Horowitz et al., Genomics, 4:87-96 (1989)). Analysis of mutations is complicated by the existence of a pseudogene which is approximately 16 kilobases (Kb) downstream from the glucocerebrosidase gene. Zimran et al., J. Clin. Invest., 86:1137-1141 (1990). The pseudogene is about 95% homologous to the functional gene. It is transcribed (Sorge et al., J. Clin. Invest., 86:1137-1141 (1990)), but cannot be translated into glucocerebrosidase because of numerous deletions of coding sequences.
Point mutations that cause Gaucher disease have been summarized recently. Latham et al., DNA Cell Biol., 10:15-21 (1991) and Grabowski et al., CRC Crit. Rev. Biochem. Mol. Biol., 25:385-414 (1990). In addition, fusion genes in which the 5' sequence is that of the active gene and the 3' sequence that of the pseudogene have been documented See, Zimran et al., J. Clin. Invest., 85:219-222 (1990); Latham et al., DNA Cell Biol., 10:15-21 (1991); Eyal et al., Gene, 96:277-283 (1990). When investigated at the genomic level, at least some such fusion genes appear to be the result of unequal crossing-over with loss of the portion of the gene between the gene and pseudogene. Zimran et al., J. Clin. Invest., 85:219-222 (1990).
The disease is most prevalent in the Jewish population with a heterozygote frequency that has been estimated to approach 9%. Zimran et al., Am. J. Hum. Genet., (1991). In Jewish patients with clinically significant Gaucher disease, about 75% of the disease-causing alleles contain a characteristic adenine to guanine (A.fwdarw.G) mutation at cDNA nucleotide position (nt) 1226 (designated the 1226G mutation) which is in the codon coding for amino acid residue 370 of the mature protein. See, Tsuji et al., Proc. Natl. Acad. Sci., U.S.A., 85:2349-2352, 5708 (1988); Zimran et al., Lancet, 2:349-352 (1989). The corresponding position of the mutation in the glucocerebrosidase gene is in exon 9 at nucleotide position 2. The same mutation is also common in the non-Jewish population, where it is found to account for approximately 25% of the disease-producing alleles. This mutation is always found in a gene that also contains a characteristic RFLP (restriction fragment length polymorphisms) with the enzyme Pvu II at genomic nt 3931, suggesting that the mutation may have occurred only once. Zimran et al., Am J. Hum. Genet., 46:902-905 (1990).
A second, much less common mutation is at cDNA nucleotide position 1448 where cytosine has been substituted for thymine (T.fwdarw.C). See, Tsuji et al., N. Engl. J. Med., 316:570-621 (1987); Dahl et al., Am. J. Hum. Genet. 47:275-278 (1990). The corresponding position of the mutation in the functional glucocerebrosidase gene is in exon 10 at nucleotide position 60. The 1448C mutation accounts for only about 2% of Jewish Gaucher disease producing alleles and for about 40% of the alleles in non-Jewish patients. Thus, in both Jewish and non-Jewish patients many of the Gaucher disease alleles have remained unidentified and have been designated "?".
The T.fwdarw.C point mutation in the functional glucocerebrosidase gene exactly matches the sequence found normally in the glucocerebrosidase pseudogene cDNA. See Horowitz et al., Genomics, 4:87-96 (1989), Tsuji et al., supra, and Sorge et al., Proc. Natl. Acad. Sci., U.S.A., 82:7289-7293 (1985). In addition, the presence of the T.fwdarw.C point mutation in exon 10 has been identified in a fusion gene which was the result of rearrangement of DNA in the glucocerebrosidase gene complex. See, Zimran et al., J. Clin. Invest., 85:219-222 (1990). The fusion gene resulted from an unequal cross-over event between the functional glucocerebrosidase gene and the pseudogene.
In this particular fusion gene, the 5' end of the transcribed cDNA was the functional gene and the 3' end was the pseudogene. The cross-over event occurred 5' or upstream to exon 10. Thus, the region of the pseudogene containing the cysteine nucleotide corresponding to the point mutation in the functional gene is in the 3' region of the fusion gene. In this situation, the nucleotide position of the cystein nucleotide would not alter. However, if an unequal cross-over event occurs sufficiently 5' to the mutation, the nucleotide position of the mutation in exon 10 may change. Therefore, the designation of nucleotide position 60 in exon 10 corresponding to nucleotide position 1448 in the cDNA would no longer be accurate. However, the region surrounding the mutation would be found in the same context, i.e., the surrounding nucleotides would be the same.
Three clinical subtypes of Gaucher Disease have been delineated. See, Beutler, Blood Rev., 2:59-70 (1988); Martin et al., Adv. Pediatr., 36:277-306 (1989). Type I is by far the most common; more than 99% of Gaucher disease patients have type I disease. It is defined by the fact that there is no neurologic involvement. Type II disease is a fulminating disorder with severe neurologic manifestations and death within the first 18 months of life Type III, the juvenile form of the disorder is characterized by later onset of neurologic symptoms than type II disease and by a chronic course.
Although all body cells are deficient in glucocerebrosidase activity in Gaucher disease, it is the glycolipid engorged macrophages that are responsible for all of the non-neurologic disease manifestation. The liver and spleen are usually enlarged. Splenomegaly results in or contributes to thrombocytopenia. Hepatic involvement is often associated with fibrosis and with abnormal liver function tests. In some patients right-to-left pulmonary shunting occurs, presumably secondary to the liver disease. Direct involvement of the pulmonary parenchyma may also rarely occur. Schneider et al., Am. J. Med., 63:475-480 (1977).
Bone involvement is common in Gaucher disease. Flaring of the distal femur, the so-called Erlenmeyer flask deformity, is a classical sign of the disease. Aseptic necrosis of the femoral heads, bone infarcts, and pathologic fractures of the long bone are all frequent complications of Gaucher disease. Stowens et al., Medicine, 64:310-322 (1985). Bone crises (Yosipovitch et al., Isr. J. Med. Sci., 26:593-595 (1990)), episodes of pain and swelling, sometimes accompanied by fever but without X-ray changes are common, recurrent manifestations of the disease.
There are patients with Type I disease who experience minimal manifestations of the disorder or none at all. Often the diagnosis in patients with such very mild disease is made in middle or old age. The presence of Gaucher disease in such patients is often appreciated only when bone marrow examination is performed for some unrelated disorder or in the course of investigation of modest thrombocytopenia. On more careful examination slight splenomegaly is often detected and minimal stigmata of the disease may be apparent when skeletal X-rays are examined. Such patients usually need no treatment.
In type II disease, neurologic findings usually become manifest in the middle of the first year of life with the development of oculomotor apraxia, strabismus, hypertonicity and retroflexion of the head. Similar neurologic symptoms occurring in the first few years of life and occasionally even later characterize type III disease.
Determination of leukocyte .beta.-glucoside activity is a reliable and simple way to diagnose Gaucher disease. Unfortunately, most patients with the disorder are still diagnosed by bone marrow examination. While this is understandable if the diagnosis was not suspected, it is an inappropriate and anachronistic procedure when Gaucher disease has been included in the differential diagnosis. Beutler and Savin, Blood, 76:646-648 (1990). Ancillary tests that are useful include the determination of the activity of serum acid phosphatase (Robinson et al., Clin. Chem., 26:371-382 (1980)) and the angiotensin converting enzyme. Lieberman et al., N. Engl. J. Med., 294:1442-1444 (1976). The levels of these enzymes, as well as levels of a number of lysosomal enzymes that are not usually measured in clinical laboratories, is increased in most but not all patients with Gaucher disease.
Recently, facile technology for the detection of the common mutations, such as those at nucleotide position 1226 (Beutler et al., Clin Chim. Acta., 194:161-166 (1990)) and nucleotide position 1448 (Zimran et al., Lancet, 2:349-352 (1989)), have been developed using the polymerase chain reaction (PCR).